Direct expression of peptides into culture media

ABSTRACT

Expression systems are disclosed for the direct expression of peptide products into the culture media where genetically engineered host cells are grown. High yield was achieved with novel vectors, a special selection of hosts, and/or fermentation processes which include careful control of cell growth rate, and use of an inducer during growth phase. Special vectors are provided which include control regions having multiple promoters linked operably with coding regions encoding a signal peptide upstream from a coding region encoding the peptide of interest. Multiple transcription cassettes are also used to increase yield. The production of amidated peptides using the expression systems is also disclosed.

RELATED APPLICATION

[0001] This application is a continuation-in-part of co-pendingprovisional U.S. patent application Ser. No. 60/043,700 filed Apr. 16,1997.

FIELD OF THE INVENTION

[0002] The present invention relates to direct expression of a peptideproduct into the culture medium of genetically engineered host cellsexpressing the peptide product. More particularly, the invention relatesto expression vectors, host cells and/or fermentation methods forproducing a peptide product that is excreted outside the host into theculture medium in high yield. In some embodiments, the invention relatesto direct expression of a peptide product having C-terminal glycinewhich is thereafter converted to an amidated peptide having an aminogroup in place of said glycine.

DESCRIPTION OF THE RELATED ART

[0003] Various techniques exist for recombinant production of peptideproducts, i.e. any compound whose molecular structure includes aplurality of amino acids linked by a peptide bond. A problem when theforeign peptide product is small is that it is often readily degradableby endogenous proteases in the cytoplasm or periplasm of the host cellthat was used to express the peptide. Other problems include achievingsufficient yield, and recovering the peptide in relatively pure formwithout altering its tertiary structure (which can undesirably diminishits ability to perform its basic function). To overcome the problem ofsmall size, the prior art has frequently expressed the peptide productof interest as a fusion protein with another (usually larger) peptideand accumulated this fusion protein in the cytoplasm. The other peptidemay serve several functions, for example to protect the peptide ofinterest from exposure to proteases present in the cytoplasm of thehost. One such expression system is described in Ray et al.,Bio/Technology, Vol. 11, pages 64-70, (1993).

[0004] However, the isolation of the peptide product using suchtechnology requires cleavage of the fusion protein and purification fromall the peptides normally present in the cytoplasm of the host. This maynecessitate a number of other steps that can diminish the overallefficiency of the process. For example, where a prior art fusion proteinis accumulated in the cytoplasm, the cells must usually be harvested andlysed, and the cell debris removed in a clarification step. All of thisis avoided in accordance with the present invention wherein the peptideproduct of interest is expressed directly into, and recovered from, theculture media.

[0005] In the prior art it is often necessary to use an affinitychromatography step to purify the fusion protein, which must stillundergo cleavage to separate the peptide of interest from its fusionpartner. For example, in the above-identified Bio/Technology article,salmon calcitonin precursor was cleaved from its fusion partner usingcyanogen bromide. That cleavage step necessitated still additional stepsto protect cysteine sulfhydryl groups at positions 1 and 7 of the salmoncalcitonin precursor. Sulfonation was used to provide protecting groupsfor the cysteines. That in turn altered the tertiary structure of salmoncalcitonin precursor requiring subsequent renaturation of the precursor(and of course removal of the protecting groups).

[0006] The peptide product of the invention is expressed only with asignal sequence and is not expressed with a large fusion partner. Thepresent invention results in “direct expression”. It is expressedinitially with a signal region joined to its N-terminal side. However,that signal region is post-translationally cleaved during the secretionof the peptide product into the periplasm of the cell. Thereafter, thepeptide product diffuses or is otherwise excreted from the periplasm tothe culture medium outside the cell, where it may be recovered in propertertiary form. It is not linked to any fusion partner whose removalmight first require cell lysing denaturation or modification, althoughin some embodiments of the invention, sulfonation is used to protectcysteine sulfhydryl groups during purification of the peptide product.

[0007] Another problem with the prior art's accumulation of the peptideproduct inside the cell, is that the accumulating product can be toxicto the cell and may therefore limit the amount of fusion protein thatcan be synthesized. Another problem with this approach is that thelarger fusion partner usually constitutes the majority of the yield. Forexample, 90% of the production yield may be the larger fusion partner,thus resulting in only 10% of the yield pertaining to the peptide ofinterest. Yet another problem with this approach is that the fusionprotein may form insoluble inclusion bodies within the cell, andsolubilization of the inclusion bodies followed by cleavage may notyield biologically active peptides.

[0008] The prior art attempted to express the peptide together with asignal peptide attached to the N-terminus to direct the desired peptideproduct to be secreted into the periplasm (see EP 177,343, GenentechInc.). Several signal peptides have been identified (see Watson, M.Nucleic Acids Research, Vol 12, No.13, pp: 5145-5164). For example,Hsiung et al. (Biotechnology, Vol 4, November 1986, pp: 991-995) usedthe signal peptide of outer membrane protein A (OmpA) of E. coli todirect certain peptides into the periplasm. Most often, peptidessecreted to the periplasm frequently tend to stay there with minimalexcretion to the medium. An undesirable further step to disrupt orpermealize the outer membrane may be required to release sufficientamounts of the periplasmic components. Some prior art attempts toexcrete peptides from the periplasm to the culture media outside thecell have included compromising the integrity of the outer membranebarrier by having the host simultaneously express the desired peptideproduct containing a signal peptide along with a lytic peptide proteinthat causes the outer membrane to become permeable or leaky (U.S. Pat.No. 4,595,658). However, one needs to be careful in the amount of lyticpeptide protein production so as to not compromise cellular integrityand kill the cells. Purification of the peptide of interest may also bemade more difficult by this technique.

[0009] Aside from outer membrane destabilization techniques describedabove there are less stringent means of permeabilizing the outermembrane of gram negative bacteria. These methods do not necessarilycause destruction of the outer membrane that can lead to lower cellviability. These methods include but are not limited to the use ofcationic agents (Martti Vaara., Microbiological Reviews, Vol. 56, pages395-411 (1992)) and glycine (Kaderbhai et al., Biotech. Appl. Biochem,Vol. 25, pages 53-61 (1997)) Cationic agents permeabilize the outermembrane by interacting with and causing damage to thelipopolysaccharide backbone of the outer membrane. The amount of damageand disruption can be non lethal or lethal depending on theconcentration used. Glycine can replace alanine residues in the peptidecomponent of peptidoglycan. Peptidoglycan is one of the structuralcomponents of the outer cell wall of gram negative bacteria. Growing E.coli in high concentration of glycine increases the frequency ofglycine-alanine replacement resulting in a defective cell wall, thusincreasing permeability.

[0010] Another prior art method of causing excretion of a desiredpeptide product involves fusing the product to a carrier protein that isnormally excreted into the medium (hemolysin) or an entire proteinexpressed on the outer membrane (e.g. ompF protein). For example, humanβ-endorphin can be excreted as a fusion protein by E. coli cells whenbound to a fragment of the ompF protein (EMBO J., Vol 4, No. 13A,pp:3589-3592, 1987). Isolation of the desired peptide product isdifficult however, because it has to be separated from the carrierpeptide, and involves some (though not all) of the drawbacks associatedwith expression of fusion peptides in the cytoplasm.

[0011] Yet another prior art approach genetically alters a host cell tocreate new strains that have a permeable outer membrane that isrelatively incapable of retaining any periplasmic peptides or proteins.However, these new strains can be difficult to maintain and may requirestringent conditions which adversely affect the yield of the desiredpeptide product.

[0012] Raymond Wong et al. (U.S. Pat. No. 5,223,407) devised yet anotherapproach for excretion of peptide products by making a recombinant DNAconstruct comprising DNA coding for the heterologous protein coupled inreading frame with DNA coding for an ompA signal peptide and controlregion comprising a tac promoter. This system reports yieldssignificantly less than those achievable using the present invention.

[0013] Although the prior art may permit proteins to be exported fromthe periplasm to the media, this can result in unhealthy cells whichcannot easily be grown to the desirable high densities, thus adverselyaffecting product yield.

[0014] The present invention seeks to produce peptide in high yield withan efficient expression vector, and to provide high yield culturingtechniques and other improvements which permits high yield recovery ofexcreted peptide of interest from the culture media, without overlydisrupting the integrity of the cell membrane.

SUMMARY OF THE INVENTION

[0015] Accordingly, it is an object of the present invention to have apeptide product accumulate in good yield in the medium in whichpeptide-producing host cells are growing. This is advantageous becausethe medium is relatively free of many cellular peptide contaminants.

[0016] It is another object of the invention to provide an improvedfermentation process for increasing the yield of a peptide productexpressed by genetically engineered host cells.

[0017] It is another object of the invention to provide geneticallyengineered host cells that are particularly useful in expressing thenovel expression vectors of the invention.

[0018] It is another object of the invention to provide a host cellwhich is particularly suited to the production of salmon calcitoninprecursor, regardless of the expression vector utilized for expressionof salmon calcitonin.

[0019] It is a further object of the invention to provide improvedmethods for the production of amidated peptides utilizing precursorpeptides having C-terminal glycines, which precursors are am idatedfollowing direct expression into the culture medium in accordance withthe invention.

[0020] In one embodiment, the invention provides an expression vectorcomprising: (a) a coding region with nucleic acids coding for a peptideproduct coupled in reading frame 3′ of nucleic acids coding for a signalpeptide; and (b) a control region linked operably with the codingregion, said control region comprising a plurality of promoters and atleast one ribosome binding site, wherein at least one of said promotersis tac. Host cells transformed or transfected with the vector areprovided, as are methods of direct expression of the peptide product byculturing such host cells.

[0021] In another embodiment, the invention provides a host celltransformed with an expression vector which comprises a gene forexpressing salmon calcitonin precursor, or calcitonin gene relatedpeptide precursor, said host cell being E. coli strain BLR; and methodsof culturing the same to obtain said precursor in the media.

[0022] In another embodiment, the invention provides a method ofproducing an amidated peptide product by producing a precursor having aC-terminal glycine using any of the vectors, hosts, or fermentationprocesses reported herein; and thereafter converting said glycine to anamino group to produce a peptide amide.

[0023] In another embodiment, the invention provides a method for directexpression of a peptide product into a culture medium comprising thesteps of: (a) culturing, in said medium, genetically engineered hostcells which express said peptide product together with a signal peptideunder conditions wherein growth of said host cells is controlled to staywithin a range of 0.05 to doublings per hour; wherein an inducer ispresent during some of said period of controlled growth; and (b)recovering said peptide product from the culture medium afterintracellular cleavage of the signal peptide.

[0024] In another embodiment, glycine is added to the medium during thecourse of direct expression fermentation, in order to increase thepermeability of the outer membrane and enhance excretion of the peptideproduct.

BRIEF DESCRIPTION OF THE FIGURES

[0025]FIGS. 1A and 1B show a schematic diagram of the construction ofthe pSCT-016B vector (1A) which is used in the construction of thepSCT-018D vector (1B) which is in turn used in the construction ofvector pSCT-025.

[0026]FIG. 2 shows a schematic diagram of the construction of thepSCT-019 vector. The LAC-OMPASCTGLY cassette used in the construction ofpSCT-025 was made by polymerase chain reaction (PCR) amplification of aportion of pSCT-019.

[0027]FIG. 3 shows a schematic diagram of the construction of thepSCT-025 vector which was used in the construction of vectors pSCT-029A,pSCT-025A, pSCT-037 and pSCT-038.

[0028]FIG. 4 shows a schematic diagram of the construction of thepSCT-029A vector which was used in the construction of vector pSCT-038and pSCT-034. In addition, pSCT-029A was used to transform E. coli BLRand produce the novel digenic UGL 165 clone.

[0029]FIG. 5 shows a schematic diagram of the construction of the pSEC-Evector which was used in the construction of vector pSEC-EY.

[0030]FIG. 6 shows a schematic diagram of the construction of the pPRLA4vector which was used in the construction of vector pSEC-EY.

[0031]FIG. 7 shows a schematic diagram of the construction of thepSEC-EY vector which was used in the construction of vector pSCT-037 andpSCT-038.

[0032]FIG. 8 shows a schematic diagram of the construction of pSCT-037and pSCT-038 vectors pSCT-037 was used to transform E. coli BLR andproduce the monogenic UGL 702 clone pSCT-038 was used to transform E.coli BLR and produce the digenic UGL 703 clone. PSCT-038 contains twocopies of the OmpA-sCTgly operon (encoding the OmpA signal together withsalmon calcitonin precursor) making it a novel digenic expressionvector. It also contains copies of genes encoding two sec machineryproteins which enhance translocation across the inner membrane into theperiplasmic space. The following is a list of abbreviations used indescribing this vector of FIG. 8:

[0033] TAC—Hybrid promoter of tryptophan E and lac operator sequences;

[0034] LAC P/O—Region containing lac promoter and lac operator of βgalactosidase gene;

[0035] LAC-IQ—Gene coding for the lac repressor that binds to operatorregion of lac promoter and tac promoter. IPTG competes with lacrepressor and inhibits binding of lac repressor to operator region ofboth tac promoter and lac promoter, thus inducing said promoters.

[0036] TRP P/O—Promoter operator region of tryptophan E gene;

[0037] OMPA-SCTGLY—Gene fusion containing secretory signal sequence ofthe outer membrane protein A gene and the coding sequence for glycineextended salmon calcitonin (the salmon calcitonin precursor);

[0038] SEC-E (also known as “PrlG”)—Gene coding for secretion factor Eof E. coli. It combines with prlA [also known as secy] or prlA-4 to formthe inner membrane translocation domain of the sec pathway by whichsignal sequence containing proteins are translocated from the cytoplasmto the periplasm;

[0039] PRLA-4—Mutant allele of prlA gene;

[0040] RRNB T1-T2—Tandem transcription terminators 1 and 2 from E. coilRibosomal protein gene; and

[0041] KAN-R—Kanamycin resistance gene.

[0042]FIG. 9 shows a schematic diagram of the PSCT-034 vector which wasused to transform E. coli BLR and produce the trigenic UGL 168 clone.

[0043]FIG. 10 shows cell growth and sCTgly production of UGL 165 clone(plasmid pSCT029A in E. coil BLR) over time after a typical 1 literfermentation in the presence of inducer. Cell growth was measured bylight absorbance at a wavelength of 600 nm. sCTgly production wasreported as mg of sCTgly excreted per liter of incubation medium. Timezero indicates the time at which inducer is first added to the culturemedium where host cells of the invention are being cultured.

[0044]FIG. 10 shows that most of the sCTgly production by UGL 165 occursbetween 20 and 25.5 hours after inducer is first added to the culturemedia where host cells of the invention are being cultured.

[0045]FIG. 11 shows cell growth and sCTgly production of UGL 703 clone(plasmid pSCT 038 in E. coil BLR) over time after a typical 1 literfermentation in the presence of inducer. Cell growth was measured bylight absorbance at a wavelength of 600 nm. sCTgly production wasreported as mg of sCTgly excreted per liter of incubation medium. Timezero indicates the time at which inducer is first added to the culturemedium where host cells of the invention are being cultured. FIG. 11shows that most of the sCTgly production by UGL 703 occurs between 20and 26 hours after culture in the presence of inducer.

[0046]FIG. 12 shows a comparison of the cell growth of UGL 172 clone(plasmid pSCT 025 in E. coil BLR), UGL 165 clone and UGL 168 clone(plasmid PSCT 034 in E. coli BLR) in a typical 1 liter fermentation overtime after incubation in the presence of inducer as measured byabsorbance at a wavelength of 600 nm. FIG. 12 shows no significantdifferences in cell growth rates of UGL 165 and UGL 172 while UGL 168shows a slight reduction in cell growth rate in this particularexperiment.

[0047]FIG. 13 shows a comparison of sCTgly production by UGL 172 clone,UGL 165 clone and UGL 168 clone over time in a typical 1 literfermentation after incubation in the presence of inducer reported as mgof sCTgly excreted per liter of incubation medium. FIG. 13 shows thatthe digenic UGL 165 clone is best suited for production of sCTgly withthe trigenic clone being second best over the monogenic UGL 173 clone.

[0048]FIGS. 14A and 14B show a comparison of sCTgly production (14A) andcell growth (14B) over time after a typical 1 liter fermentation in thepresence of inducer by UGL 165 clone and either in the presence orabsence of oxygen supplementation to the air feed. Cell growth wasmeasured as g of wet cell weight per liter of incubation media. sCTglyproduction was reported as mg of sCTgly excreted per liter of incubationmedium. FIGS. 14A and 14B show that added oxygen in the fermentationmedium is not critical to cell growth of UGL 165 but is very importantin increasing the production of sCTgly.

[0049]FIGS. 15A and 15B show a comparison of the cell growth (15A) andsCTgly production (15B) over time after a typical 1 liter fermentationin the presence of inducer by the E. coli strains WA837 and BLR whereeach strain is expressing the pSCT-029A vector (UGL164 and UGL165respectively). Cell growth was measured by light absorbance at awavelength of 600 nm. sCTgly production was reported as mg of sCTglyexcreted per liter of incubation medium FIGS. 15A and 15B show that theBLR E. coli strain is more suited for sCTgly production than the WA837E. coli strain.

[0050]FIG. 16 shows a comparison of sCTgly production after a typical 1liter fermentation in the presence of inducer by the E. coli strainsWA837, BLR, BL21 and B834, where each strain is expressing the PSCT-029Avector (UGL164, UGL165, UGL167 and UGL166 respectively). sCTglyproduction was reported as mg of sCTgly excreted per liter of incubationmedium. FIG. 16 shows that the E. coli BLR strain is more suited forsCTgly production than each of the WA837, BL21 and B834 E. coli strains.

[0051]FIG. 17 shows a comparison of the best sCTgly production observedfrom different experiments after 1 liter fermentations in the presenceof inducer by UGL 165 (pSCT-029A in BLR), UGL 168 (pSCT-034 in BLR), UGL172 (pSCT-025 in BLR), UGL 702 (pSCT-037 in BLR) and UGL 703 (pSCT-038in BLR) clones. sCTgly production was reported as mg of sCTgly excretedper liter of incubation medium. FIG. 17 shows that the digenic UGL 703and UGL 165 clones are more suited for sCTgly production than themonogenic UGL 172 and UGL 702 clones and the trigenic UGL 168 clone.FIG. 17 also shows that the digenic UGL 703 clone that expressessecretion factors is more suited for sCTgly production than the digenicUGL 165 clone which does not express secretion factors.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention permits peptide product yields in excess of100 mg per liter of media. It does so with novel expression vectors,novel hosts (as transformed, transfected or used in accordance with theinvention), novel fermentation processes, or a combination of two ormore of the foregoing.

[0053] Overview of a Preferred Expression Vector

[0054] In one embodiment, the present invention provides an expressionvector which comprises a coding region and a control region. The codingregion comprises nucleic acids for a peptide product of interest coupledin reading frame downstream from nucleic acids coding for a signalpeptide. The control region is linked operably to the coding region andcomprises a plurality of promoters and at least one ribosome bindingsite, wherein at least one of the promoters is selected from the groupconsisting of tac and lac.

[0055] Preferably, the vector comprises a plurality of transcriptioncassettes placed in tandem, each cassette having the control region andthe coding region of the present invention. Such a digenic vector ormultigenic vector is believed to provide better expression than would adicistronic or multicistronic expression vector This is a surprisingimprovement over dicistronic or multicistronic expression which is notbelieved to be suggested by the prior art.

[0056] The vector can optionally further comprise nucleic acids codingfor a repressor peptide which represses operators associated with one ormore of the promoters in the control region, a transcription terminatorregion, a selectable marker region and/or a region encoding at least onesecretion enhancing peptide. Alternatively, in some embodiments, nucleicacids coding for a repressor peptide and a secretion enhancing peptidemay be present on a separate vector co-expressed in the same host cellas the vector expressing the peptide product.

[0057] Specific examples of constructed expression vectors, and methodsfor constructing such expression vectors are set forth intra. Manycommercially available vectors may be utilized as starting vectors forthe preferred vectors of the invention. Some of the preferred regions ofthe vectors of the invention may already be included in the startingvector such that the number of modifications required to obtain thevector of the invention is relatively modest. Preferred starting vectorsinclude but are not limited to pSP72 and pKK233-2.

[0058] It is believed that the novel vectors of the invention impartadvantages which are inherent to the vectors, and that those unexpectedadvantages will be present even if the vectors are utilized in hostcells other than the particular hosts identified as particularly usefulherein, and regardless of whether the improved fermentation processdescribed herein is utilized.

[0059] Likewise, in certain embodiments, particular host cells areidentified as being particularly useful in the expression of peptidessuch as salmon calcitonin precursor and calcitonin gene related peptideprecursor. The advantages imparted by specifically utilizing theparticular host cells identified herein are believed to exist regardlessof whether the expression vector is one of the novel vectors describedherein or whether the novel fermentation process described herein isutilized. In other words, it is believed that these host cells willimpart significant unexpected advantages even utilizing prior artfermentation or prior art vectors.

[0060] The novel fermentation process is believed to provide increasedyield because of inherent advantages imparted by the fermentationprocess. It is believed that these advantages will be present regardlessof whether the preferred host cells and/or novel vectors describedherein are utilized.

[0061] Notwithstanding the foregoing, one preferred embodiment of theinvention simultaneously utilizes the improved expression vectors of theinvention transformed into the particularly identified host cells of theinvention and expressed utilizing the preferred fermentation inventiondescribed herein. When all three of these inventions are used incombination, it is believed that a significant enhancement of yield andrecovery of product can be achieved relative to the prior art.

[0062] The Control Region

[0063] The control region is operably linked to the coding region andcomprises a plurality of promoters and at least one ribosome bindingsite, wherein at least one of the promoters is selected from the groupconsisting of lac and tac. It has surprisingly been found that theforegoing combination of promoters in a single control regionsignificantly increases yield of the peptide product produced by thecoding region (as described in more detail intra). It had been expectedthat two such promoters would largely provide redundant function, andnot provide any additive or synergistic effect. Experiments conducted byapplicants have surprisingly shown a synergy in using the claimedcombination of promoters. Other promoters are known in the art, and maybe used in combination with a tac or lac promoter in accordance with theinvention. Such promoters include but are not limited to lpp, ara B,trpE, gal K.

[0064] Preferably, the control region comprises exactly two promoters.When one of the promoters is tac, it is preferred that the tac promoterbe 5′ of another promoter in the control region. When one of thepromoters is lac, the lac promoter is preferably 3′ of another promoterin the control region. In one embodiment, the control region comprisesboth a tac promoter and a lac promoter, preferably with the lac promoterbeing 3′ of the tac promoter.

[0065] The Coding Region

[0066] The coding region comprises nucleic acids coding for a peptideproduct of interest coupled in reading frame downstream from nucleicacids coding for a signal peptide whereby the coding region encodes apeptide comprising, respectively, from N terminus to C terminus thesignal and the peptide product. Without intending to be bound by theory,it is believed that the signal may provide some protection to thepeptide product from proteolytic degradation in addition toparticipating in its secretion to the periplasm.

[0067] Many peptide signal sequences are known and may be used inaccordance with the invention. These include signal sequences of outermembrane proteins of well-characterized host cells, and any sequencescapable of translocating the peptide product to the periplasm and ofbeing post-translationally cleaved by the host as a result of thetranslocation. Useful signal peptides include but are not limited to OmpA, pel B, Omp C, Omp F, Omp T, β-la, Pho A, Pho S and Staph A.

[0068] The peptide product is preferably small enough so that, absentthe present invention, it would usually require a fusion partner usingprior art technology. Typically, the peptide product has a molecularweight of less than 10 KDa. More preferably, the peptide product has aC-terminal glycine, and is used as a precursor to an enzymatic amidationreaction converting the C-terminal glycine to an amino group, thusresulting in an amidated peptide. Such a conversion is described in moredetail infra. Numerous biologically important peptide hormones andneurotransmitters are amidated peptides of this type. For example, thepeptide product coded by the coding region may be salmon calcitoninprecursor or calcitonin gene related peptide precursor, both of whichhave C-terminal glycines and both of which may be enzymatically amidatedto mature salmon calcitonin or mature calcitonin gene related peptide.Other amidated peptides that may be produced in accordance with theinvention include but are not limited to growth hormone releasingfactor, vasoactive intestinal peptide and galanin. Other amidatedpeptides are well known in the art.

[0069] Analogs of parathyroid hormone could also be produced inaccordance with the invention. For example, a peptide having the first34 amino acids of parathyroid hormone can provide a function similar tothat of parathyroid hormone itself, as may an amidated version of the 34amino acid analog. The latter may be produced by expressing, inaccordance with one or more of the expression systems and methodsdescribed herein, the first 34 amino acids of parathyroid hormone,followed by glycine-35. Enzymatic amidation as disclosed herein couldthen convert the glycine to an amino group.

[0070] While preferred embodiments of the direct expression systemdescribed herein produce peptides having C-terminal glycine, it isbelieved that any peptide will enjoy good yield and easy recoveryutilizing the vectors, hosts and/or fermentation techniques describedherein.

[0071] Other Optional Aspects of a Preferred Vector of the Invention orof Other Vectors to be Expressed in the Same Host as the Vector of theInvention

[0072] Repressor

[0073] Optionally, the preferred vector of the present invention maycontain nucleic acids coding for a repressor peptide capable ofrepressing expression controlled by at least one of the promoters.Alternatively, however, the nucleic acids coding for a repressor peptidemay be present on a separate vector in a host cell with the vector ofthe present invention. Appropriate repressors are known in the art for alarge number of operators. Preferably, the nucleic acids coding for therepressor encode a lac repressor in preferred embodiments of theinvention because it represses the lac operator that is included withboth tac and lac promoters, at least one of which promoters is alwayspresent in preferred vectors of the invention.

[0074] Selectable Marker

[0075] It is preferred that any of a large number of selectable markergenes (e.g. a gene encoding kanamycin resistance) be present in thevector of the present invention. This will permit appropriate specificselection of host cells that are effectively transformed or transfectedwith the novel vector of the invention.

[0076] Secretion Enhancing Peptide

[0077] Nucleic acids coding for at least one secretion enhancing peptideare optionally present in the vector of the present invention.Alternatively, the nucleic acids coding for a secretion enhancingpeptide may be present on a separate vector expressed in the same hostcell as the vector encoding the peptide product. Preferably, thesecretion enhancing peptide is selected from the group consisting ofSecY (prlA) or prlA-4. It is pointed out that SecY and prlA areidentical, the two terms being used as synonyms in the art. prlA-4 is aknown modification of prlA and has a similar function. Another preferredsecretion enhancing peptide is SecE also known as “prlG”, a term used asa synonym for “SecE”. Most preferably, a plurality of secretionenhancing peptides are encoded, at least one of which is SecE and theother of which is selected from the group consisting of SecY (prlA) andprlA-4. The two are believed to interact to aid translocation of thepeptide product from cytoplasm to periplasm. Without intending to bebound by theory, these secretion enhancing peptides may help protect thepeptide product from cytoplasmic proteases in addition to theirsecretion enhancing functions.

[0078] Host Cell

[0079] The present invention also provides a host cell transformed ortransfected with any of the vectors of the present invention.Preferably, the host cell is a bacterial cell. More preferably, the hostcell is a gram negative bacterial cell. Still more preferably, the hostcell is E. coli. More preferably, the E. coli is strain BLR, BL21 orWA837. Most preferably, the E. coli is strain BLR. Also most preferably,the host cell further expresses at least one secretion-enhancingpeptide.

[0080] The present invention further provides a host cell transformedwith an expression vector which comprises a gene for expressing salmoncalcitonin precursor or Calcitonin Gene Related Peptide precursor, saidhost cell being E. coli strain BLR. It is believed that BLR expressionof these two peptides will be particularly effective even where priorart vectors are used for the expression. In other words, it is notbelieved that the novel expression vectors reported herein are requiredfor good expression of these two peptides in a BLR host.

[0081] Method of Producing a Heterologous Peptide

[0082] Novel fermentation conditions are provided for growing host cellsto very high cell densities under culture conditions which permit thediffusion or excretion of the peptide product into the culture medium inhigh yield.

[0083] Host cells useful in the novel fermentation include but are notlimited to the host cells discussed supra, and/or host cells transformedor transfected with one or more of the novel expression vectorsdiscussed supra. Other host cells genetically engineered to expresspeptide product together with a signal region may be used. The cells areplaced in a fermenter which preferably includes appropriate means offeeding air or other gases, carbon source, and other components to themedia and means for induction of the promoter Appropriate means formonitoring oxygen content, cell density, pH and the like are alsopreferred.

[0084] Applicants have found that significantly improved yield ofpeptide product directly expressed into the culture medium is obtainedby carefully controlling the average cell growth rate within a criticalrange between 0.05 and 0.20 doublings per hour. It is preferred thatthis controlled growth begin in early lag phase of the culture. It ismore preferable to maintain average cell growth rate during thefermentation period (i.e. the period during which growth is beingcontrolled as set forth herein), between 0.10 and 0.15 doublings perhour, most preferably 0.13 doublings per hour. Growth rate may becontrolled by adjusting any of the parameters set forth infra in thesection entitled “Production of sCTgly (Fermentation)”, specifically theformula equating the feed rate “Q” to numerous other parameters.Applicants have found that varying the rate of carbon source being fedto the fermenting cells is an advantageous method of maintaining thegrowth rate within the critical range. In order to maintain the growthrate relatively constant, the amount of carbon source feeding into thefermenter tends to increase proportionally to the growth in number ofcells.

[0085] Applicants have also discovered that significantly improved yieldcan be obtained by providing inducer during said fermentation period ofcontrolled growth. Like carbon source, feeding proper amounts of inducerinvolves increasing the rate of feed proportional to growth in number ofcells. Since both carbon source and inducer feed preferably increase ina manner which is linked to cell growth, applicants have found that itis advantageous to mix feed and inducer together and to feed the mixtureof the two at the appropriate rate for controlling cell growth (with thecarbon source), thus simultaneously maintaining a continuous feed ofinducer which stays at a constant ratio relative to the amount of carbonsource. However, it is of course possible to feed carbon source andinducer separately. Even then, however, if a chemical inducer that maybe toxic to the cells in large amounts is used, it is desirable that theinducer and carbon source be added during each hour of culturing inamounts such that the weight ratio of the inducer added in any givenhour to the carbon source added in that same hour does not vary by morethan 50% from the ratio of the amount of inducer added during theentirety of the fermentation process (controlled growth period) toamount of carbon source added during the entirety of the fermentationprocess. The 50% variance is measured from the lower ratio of two ratiosbeing compared. For example, where the ratio of carbon source to inducerfor the entire fermentation is 2 to 1, the ratio in any given hour ispreferably no higher than 3 to 1 and no lower than 1.333 to 1. It isalso possible to induce one or more of the promoters during growth byother means such as a shift in temperature of the culture or changingthe concentration of a particular compound or nutrient.

[0086] When external carbon source feed is used as the method ofcontrolling cell growth, it is useful to wait until any carbon sourcesinitially in the media (prior to external carbon feed) have beendepleted to the point where cell growth can no longer be supportedwithout initiating external carbon feed. This assures that the externalfeed has more direct control over cell growth without significantinterference from initial (non-feed) carbon sources. An oxygen source ispreferably fed continuously into the fermentation media with dissolvedoxygen levels being measured. An upward spike in the oxygen levelindicates a significant drop in cell growth which can in turn indicatedepletion of the initial carbon source and signify that it is time tostart the external feed.

[0087] It has been unexpectedly found that peptide product yieldincreases as oxygen saturation of the fermentation media increases. Thisis true even though lower oxygen saturation levels are sufficient tomaintain cell growth. Thus, during the entire fermentation process, itis preferred that an oxygen or oxygen enriched source be fed to thefermentation media, and that at least 20% and preferably at least 50%oxygen saturation be achieved. As used herein, “oxygen saturation” meansthe percentage of oxygen in the fermentation medium when the medium iscompletely saturated with ordinary air. In other words, fermentationmedia saturated with air has an “oxygen saturation” of 100%. While it isdifficult to maintain oxygen saturation of the fermentation mediumsignificantly above 100%, i.e. above the oxygen content of air, this ispossible, and even desirable in view of higher oxygen content providinghigher yields. This may be achieved by sparging the media with gaseshaving higher oxygen content than air.

[0088] Significant yield improvement may be achieved by maintainingoxygen saturation in the fermentation medium at no lower than 70%,especially no lower than 80% Those levels are relatively easy tomaintain.

[0089] Faster agitation can help increase oxygen saturation. Once thefermentation medium begins to thicken, it becomes more difficult tomaintain oxygen saturation, and it is recommended to feed gases withhigher oxygen content than air at least at this stage. Applicants havefound that ordinary air can be sufficient to maintain good oxygensaturation until relatively late in the fermentation period. Applicantshave supplemented the air feed with a 50% oxygen feed or a 100% oxygenfeed later in the fermentation period. Preferably, the host cell iscultured for a period between 20 and 32 hours (after beginningcontrolled growth), more preferably between 22 and 29 hours, mostpreferably for about 24-27 hours.

[0090] Preferably, the host cells are incubated at a temperature between20 and 35° C., more preferably between 28 and 32° C., more preferablybetween 29.5 and 30.5° C. A temperature of 30° C. has been found optimalin several fermentations conducted by applicants.

[0091] Preferably, the pH of the culturing medium is between 6.0 and7.5, more preferably between 6.6 and 7.0, with 6.78-6.83 (e.g. 6.8)being especially preferred.

[0092] In preferred embodiments, fermentation is carried out using hoststransformed with an expression vector having a control region thatincludes both a tac and a lac promoter and a coding region includingnucleotides coding for a signal peptide upstream of nucleotides codingfor salmon calcitonin precursor. Such an expression vector preferablyincludes a plurality, especially two, transcription cassettes in tandem.As used herein, the term “transcription cassettes in tandem” means thata control and coding region are followed by at least one additionalcontrol region and at least one additional coding region encoding thesame peptide product as the first coding region. This is to bedistinguished from the dicistronic expression in which a single controlregion controls expression of two copies of the coding region. Thedefinition will permit changes in the coding region that do not relateto the peptide product, for example, insertion, in the secondtranscription cassette, of nucleotides coding a different signal peptidethan is coded in the first transcription cassette.

[0093] Numerous carbon sources are known in the art. Glycerol has beenfound effective. Preferred methods of induction include the addition ofchemical inducers such as IPTG and/or lactose. Other methods such astemperature shift or alterations in levels of nutrient may be used.Other induction techniques appropriate to the operator or the promoterin the control region (or one of the plurality of promoters being usedwhere more than one appears in the control region) may also be used.

[0094] It is typical that production of peptide product dropssignificantly at about the same time that growth of the cells in thefermentation media becomes unsustainable within the preferred growthrate discussed supra At that point, fermentation is stopped, carbonsource and inducer feed and oxygen flow are discontinued. Preferably,the culture is quickly cooled to suppress activity of proteases and thusreduce degradation of the peptide product. It is also desirable tomodify pH to a level which substantially reduces proteolytic activity.When salmon calcitonin precursor is produced using preferred vectors andhost cells of the invention, proteolytic activity decreases as pH islowered. This acidification preferably proceeds simultaneously withcooling of the media. The preferred pH ranges are discussed in moredetail infra. The same assay as is being used for measuring fermentationproduct can be used to measure degradation at different pH levels, thusestablishing the pH optimum for a given peptide and its impurities.

[0095] Recovery of the Heterologous Peptide

[0096] The present invention further provides a method for recoveringthe peptide product which comprises separating the host cells from theculture medium and thereafter subjecting the culturing medium to atleast one type of chromatography selected from the group consisting ofgel filtration, ion-exchange (preferably cation exchange when thepeptide is calcitonin), reverse-phase, affinity and hydrophobicinteraction chromatography. In a peptide containing cysteine residues,S-sulfonation may be carried out prior to or during the purificationsteps in order to prevent aggregation of the peptide and therebyincrease the yield of monomeric peptide. Preferably, threechromatography steps are used in the following order: ion exchangechromatography, reverse-phase chromatography and another ion exchangechromatography.

[0097] After fermentation is completed, the pH of the culture medium isoptionally altered to reduce the proteolytic activity. The assay used tomeasure product production can also be used to measure productdegradation and to determine the best pH for stability. Where salmoncalcitonin precursor is produced in accordance with the invention, a pHbetween 2.5 and 4.0 is preferred, especially between 3.0 and 3.5. ThesepH ranges also are believed to aid retention of salmon calcitoninprecursor on cation exchange columns, thus providing better purificationduring a preferred purification technique described herein.

[0098] Also optionally, the temperature of the medium, afterfermentation is completed, is lowered to a temperature below 10° C.,preferably between 3° C. to 5° C., most preferably 4° C. This is alsobelieved to reduce undesirable protease activity.

[0099] The present invention further provides a method of producing anamidated peptide product comprising the steps of: culturing, in aculture medium, any of the host cells of the present invention whichexpress a peptide product having a C-terminal glycine; recovering saidpeptide product from said culture medium; amidating said peptide productby contacting said peptide product with oxygen and a reducing agent inthe presence of peptidyl glycine α-amidating monooxygenase, or peptidylglycine α-hydroxylating monooxygenase. If peptidyl glycine α-amidatingmonooxygenase is not used hereinabove, and if the reaction mixture isnot already basic, then increasing pH of the reaction mixture until itis basic. Amidated peptide may thereafter be recovered from the reactionmixture preferably utilizing the purification technique described infrain Example 6.

[0100] Preferably, the host cell is cultured in a culture medium in thepresence of an inducer, while maintaining an average cell growth rateduring culturing between 0.05 and 0.20 doublings per hour.

[0101] Experimental Details

[0102] pSCT-037 and DSCT-038 Cloning Strategy

[0103] The construction of pSCT-038 and pSCT-037 is comprised of eightparts converging to create the intermediate vectors needed to constructthe final desired expression plasmids. All genes and fragments that wereused or constructed outside of the text description for this project arelisted in Table 1.

[0104] Part I Construction of pSCT-018D

[0105] The TAC promoter cartridge (Table 1) was subcloned intopGEM11ZF+(Table 1) as a Hind III-Bam HI fragment creating PGEM11ZF+TAC.The pelB-sCTgly cas2 gene (Table 1) was ligated downstream of the tacpromoter (in pGEMllZF+TAC) into the Bam HI site to create the expressionvector pSCT-013B. The tac-pelBsCTgly operon was cut from pSCT-013B usingHind III and Eco RI. This fragment was then ligated, along with a HindIII-Pst I adapter, into pSP72 (Table 1) creating pSCT-015B. Thekanamycin resistance gene was then ligated into the Pst I site ofpSCT-015B, creating pSCT-016B. The 5′ coding and control region of theβ-lactamase gene (ampicillin resistance) was deleted by cutting thevector with Pvu II and Fsp I followed by religation creating pSCT-017B.The T1-T2 transcription terminator from pSP72 T1-T2 (Table 1) was thencut and ligated into PSCT-017B using Sal I and Bgl II sites creatingpSCT-018B. The LAC-IQ gene (Table 1) containing Bam HI and Eco RI siteswas then ligated into the Bgl II-Eco RI sites of PSCT-018B creatingpSCT-018D (see FIGS. 1A and 1B)

[0106] Part II Construction of pSCT-019

[0107] The lac P/O (Table 1) with sites Pst I and Hind III and a HindIII-Eco RI fragment from pSCT-013B (containing tac-pelBsCTgly) wereinserted, in a three way ligation, into the Pst I and Eco RI sites ofpSP72 creating pSCT-015A. As described in the construction of pSCT-017B,the Kan-R gene was inserted and the β-lactamase gene was removed frompSCT-015A to create pSCT-016A and pSCT-017A. The lpp-lac-ompAsCTgly(Table I) Pst I-Acc I fragment (17 base pairs of the 5′ coding sequenceof sCTgly are also present) was cut from pSP72-OMPA and ligated into thecompatible sites in pSCT-017A to create pSCT-019 (see FIG. 2).

[0108] Part III Construction of pSCT-025

[0109] A Bam HI fragment of the PCR amplified operon lac-ompAsCTgly(Table 1) was ligated into the Bam HI site of pSCT-018D, replacing thepelBsCTgly gene creating pSCT-023(-). The (-) signifies that the insertwas inserted in the reverse orientation in relation to the tac promoter.The lac-ompAsCTgly operon was then cut from pSCT-023(-), using Bam HIand Sal I, then ligated into the compatible sites of pSCT-017B creatingpSCT-017 DELTA. The larger Bgl I-Bgl II fragment containing thekanamycin resistance gene, tac-lac promoters and ompAsCTgly was cut frompSCT-017 DELTA and ligated into the Bgl I-Bam HI sites of pSCT-018Dcreating pSCT-025 (see FIG. 3).

[0110] Part IV Construction of pSCT-029A

[0111] A PCR product of RRNB T1-T2-02 (Table 1), containing Xho I andEco RI sites, was ligated into the Sal I-Eco RI sites of pSCT-025creating pSCT-025A. A Hind III-Sac I fragment cut from pSCT-025,containing the tac-lac-ompAsCTgly RRNB T1-T2 terminators gene cartridge,was ligated into the compatible sites of pSP72 creating pCPM-01. ThepCPM-01A vector was constructed using the same method described forpSCT-025A. The pSCT-025A and pCPM-01A plasmids differ from the pSCT-025and pSCT-01 plasmids in the types of restriction sites present upstreamand downstream of the RRNB T1-T2 transcription terminator. An Xho I-EcoRI fragment, containing the tac-lac-ompAsCTgly RRNB T1-T2 terminatorsgene cartridge was cut from pCPM-01A and ligated into the Sal I-Eco RIsites of pSCT-025A creating the digenic expression vector pSCT-029A(FIG. 4). The methods used to create pSCT-029A can be repeated to createadditional polygenic expression vectors, as shown below for theconstruction of pSCT034.

[0112] Part V Construction of pSEC-E

[0113] The vector pCPM-01 was digested with Pst I and Xba I to excisethe lac P/O, which was then ligated into the compatible sites of pSP72.The PCR amplified secE gene (Table I) containing Xba I and Bam HIcloning sites was ligated into the compatible sites of the ompAsCTglyT1-T2 cistron cloning vector (Table 1) creating pCM-SECE. The secE andT1-T2 terminators were cut from PCM-SECE as an Xba I-Eco RI fragment andligated along with an Xba I-Sal I fragment, containing the lac P/O (cutfrom pSP72-lac), into the Sal I-Eco RI sites of pSP72 creating pSEC-E(see FIG. 5)

[0114] Part VI Construction of pPRLA-4 (prlA-4 is a Mutant Allele of theprLA or secY Gene)

[0115] The T1-T2 terminator region of the ompAsCTgly T1-T2 cistronvector was excised using Bam HI and Sma I, which was then ligated intothe Pvu II and Bam HI sites of pSP72 creating the PRLA4-INT intermediatecloning vector. The lac P/O was cut from pSP72-LAC with Xba I and Sac Iand ligated along with the prlA-4 PCR fragment (Table I), containing XbaI and Bam HI restriction sites, into the Sac I-Bam HI sites of PRLA4-INTcreating pPRLA-4 (see FIG. 6).

[0116] Part VII Construction of pSEC-EY

[0117] A synthetic oligonucleotide fragment containing the trpE P/Osequence (Table I) was subcloned into the Xho I-Xba I sites of pCPM-01Acreating pCPM-08. The prlA-4 and T1-T2 sequences were cut from pPRLA-4with Xba I and Xho I and ligated with a Sal I-Eco RI fragment frompSEC-E, containing the lac-secE-T1-T2 operon, into the Xba I and Eco RIsites of pCPM-08 creating pSEC-EY (see FIG. 7).

[0118] Part VIII Construction of pSCT-037 and pSCT-038

[0119] The secE and prlA-4 coding region were cut from pSEC-EY with XhoI and Bgl II. The resulting fragment was then ligated into the Sal I-BglII sites of pSCT-025A and pSCT-029A creating pSCT-037 and pSCT-038respectively (see FIG. 8)

[0120] Construction of pSCT-034

[0121] pSCT-034 is a trigenic expression plasmid containing three copiesof the Tac-Lac-ompAsCTgly RRNB T1-T2 transcription cassette. This vector(see FIG. 9) was constructed by inserting the described cartridge frompCPM-OlA into Sal I and Eco RI sites of pSCT-029A adding a third copy ofthe expression cartridge. The method of construction is identical to themethod for the construction of pSCT-029A from pCPM-OlA and pSCT-025A.The 3′ Sal I and Eco RI sites are recreated, providing the sitesnecessary for adding more copies of the cartridge. TABLE 1 Cloningfragment Component type Origin or template pGEM 11 ZF(+) Plasmid PromegaPGEX 1N Plasmid Pharmacia Biosciences pSP 72 Plasmid Promega Tacpromoter tac promoter Pharmacia Biosciences DNA block pelB-sCTgly-cas2*PCR amplified pelB-sCTgly gene assembled gene from syntheticoligonucleotides Kanamycin Gene block Pharmacia Biosciences resistancegene RRNB T1-T2 PCR fragment Ribosomal protein gene T1 and T2transcription terminators from pKK 233-2 pSP72 T1-T2 Subclone Subclonedfrom above PCR fragment into pSP72 Lac repressor PCR amplified pGEX 1-Nplasmid (LAC-IQ) gene Lac promoter/ PCR amplified pGEM11ZF + operator(P/O) fragment 1pp-lac-cmpAsCTgly PCR amplified pIN IIIA (partial)product (3′ PCR primer contains first 17 nucleotides of sCTgly) pSP72-ompA Subclone of Subcloned from above PCR above fragment fragmentinto pSP72 lac-ompAsCTgly PCR amplified pSCT-019 operon RRNB T1-T2-02PCR amplified pSCT-025 product OMPASCTGLY PCR amplified pSCT-025(template) T1-T2 CISTRON product of ompAsCTgly subcloned into pSP72T1-T2 SEC E PCR amplified E. coil WA 827 genomic gene DNA PRLA-4 PCRamnlified E. coil pr1A 4 gene from gene vector pRLA41++ TRP P/OAssembled E. coil tryptophan E synthetic promoter/operator Sequenceoligonucleotide from literature gene

[0122] Transformation of E. coli BLR with pSCT-029A or pSCT-038

[0123] After constructing the pSCT-029A or pSCT-038 plasmids, i.e.,ligation of the various DNA fragments, it is necessary to use the finalligation mixture to transform an E. coli host strain for propagation ofthe plasmid and for future protein expression work. To perform thistransformation it is necessary to cause the E. coli cells to becompetent to receive the DNA. The preparation of competent cells can bedone by a variety of methods such as CaCl₂ treatment and electroporationFor the final preparation of preferred cell lines, UGL 165 and UGL 703,we use both methods in series according to the following protocols.

[0124] I. Primary Transformation into the E. coli K-12 host, BB4

[0125] This first transformation is not essential, but is preferredbecause the E. coil BB4 K-12 host has a high transformation efficiencywhich results in a large number of transformants and a high probabilityof identifying a variety of desired clones.

A. Preparation of Competent BB4 Cells by CaCl₂ Treatment

[0126] BB4 Genotype . . . LE392.32 [F′ lacI^(Q)ZΔMI5proAB Tn 10(Tet^(R))]

[0127] 1. Prepare an overnight saturated culture of the host cell.

[0128] 2. Prepare a fresh host cell culture by inoculating 100 ml ofmedium to 0.5% (v/v) and grow to an A₆₀₀ nm of 0.02-0.03.

[0129] 3. Grow the culture to an A₆₀₀ nm of 0.15-0.3, approximately 3doublings.

[0130] 4. Store the cells on ice for 10 minutes.

[0131] 5. Remove the cells from the culture media by centrifugation, 5Krpm× 10 min.

[0132] 6. Resuspend the pelleted cells in 0.5 volumes of ice cold 0.1 MCaCl₂, store on ice for 30 minutes, then pellet the cells as previouslydescribed.

[0133] 7. Resuspend the pelleted cells in 0.1× volume of 0.1 M CaCl₂;store on ice for 1 hour prior to use.

[0134] B. Transformation Protocol

[0135] 1. To 100 ul of competent cells prepared as described add 1-2 ulof a ligation mixture which should ideally contain 2-10 ng of plasmidvector DNA.

[0136] 2. Store the mixture on ice for 30 minutes.

[0137] 3. Heat shock the mixture by placing in a heating block or waterbath at 37° C.

[0138] 4. Add to the mixture 1 ml of prewarmed culture medium andincubate the mixture at 37° C. for 30-60 minutes.

[0139] 5. Spread an appropriate amount of transformation mixture onto anappropriate solid media containing the necessary selective antibioticand incubate the plates 18-24 hours until colonies appear.

[0140] II. Secondary Transformation

[0141] Transformants are identified by a variety of methods. Severalclones are chosen for transfer to the second host, E. coli B hoststrain, BLR. BLR is the host strain of choice or fermentation andprotein expression.

[0142] The genotype of BLR is F⁻ ompT hsds_(B)(_(rB) ⁻.m_(B) ⁻) gal dcmΔ(srl-recA) 306::Tn10(Tc^(R)). E. coli cells will accept DNA after beingexposed to an electric field under controlled and specified conditions.E. coli B host strains are more easily transformed with an intactplasmid, rather than a ligation mixture, and are more receptive toforeign DNA when made competent by electroporation than by CaCl₂treatment.

[0143] A. Preparation of Competent E. coli BLR Cells for SubsequentElectroporation

[0144] 1. Prepare an overnight saturated culture of the host cell.

[0145] 2. Prepare a fresh host cell culture by inoculating 100 ml ofmedium to 1.0% (v/v) and grow to an A₆₀₀ nm of 0.3-0.5.

[0146] 3. Harvest the cells by centrifugation after chilling the cultureon ice for 15 min.

[0147] 4. Decant the supernatant, removing as much media as possible.Resuspend the cells in a total volume of 100 mls of ice cold aqueous 10%glycerol, w/v (the glycerol should be of extra high quality).Re-centrifuge the resuspended cells immediately.

[0148] 5. Resuspend the cells in 50 mls of ice cold 10% glycerol (w/v).Re-centrifuge.

[0149] 6. Resuspend the cells in 25 mls of ice cold 10% glycerol (w/v).Re-centrifuge.

[0150] 7. Repeat step 6.

[0151] 8. Resuspend the cells in a final volume of 2 mls of ice cold 10%glycerol (w/v). The final cell concentration should be 1-3×10¹⁰cells/ml. The cells can be stored for up to 1 year at −80° C.

[0152] B. Transformation by Electroporation

[0153] 1. Incubate sterile cuvettes and the white chamber slide for 10min on ice. Also incubate several polypropylene tubes on ice.

[0154] 2. Mix 40 μl of cell suspension with 1-2 μl of solutioncontaining plasmid DNA in Tris/EDTA at an approximate concentration of100 pg/μl. (The DNA mixture must be as salt free as possible to preventarcing of the equipment.) Mix the solution well and incubate on ice for0.5-1.0 min.

[0155] 3. Set the Gene-Pulser apparatus at 25 uF. Set the pulsecontroller resistance at 200 ohms. Set the Gene-Pulser apparatus to 2.5Kv with 0.2 cm cuvettes or 1.5-1.8 Kv when using 1.0 cm cuvettes.Transfer the mixture of cells and DNA to a cold electroporation cuvetteand shake the suspension to the bottom of the cuvette removing all ofthe air bubbles. Place the cuvette in the chilled safety chamber slideand push the slide into the chamber until the cuvette is seated betweenthe contacts in the base of the chamber.

[0156] 4. Pulse the cuvette once at above setting.

[0157] 5. Immediately add 1 ml of SOC (20 g Tryptone, 5 g yeast extract,0.5 g NaCl, 1 L H₂O and 20 mM glucose) buffered media to the cuvette,resuspend the cells.

[0158] 6. Transfer the suspension to a sterile 17×100 mm polypropylenetube and incubate at 37° C. for 1 hour.

[0159] 7. Plate the electroporated mixture on selective media plates.

[0160] Production of sCTglv (Fermentation)

[0161] Fermentation batch media is prepared using the components listedin Table 2. The fermentation is inoculated with a late log phase culturegrown in inoculation media (Table 2). The inoculation volume isdetermined by the amount of inoculum (number of cells) needed to reachan initial A 600 nm within the fermenter between 0.015 and 0.24. The pH,DO₂, and temperature parameters for the fermentation run are listed inTable 3. The fed batch state of the fermentation is started when theglycerol in the batch media is depleted. [Glycerol depletion can bedetermined by a spike in dissolved oxygen and/or a glycerol assay.] Thefeed rate is set to maintain a constant cell division rate based on cellmass (dry cell weight) at the time of glycerol depletion. Feedcomponents are listed in Table 2. The feed rate is based on thefollowing formula:$Q = \frac{(V) \times ({MU}) \times ({dcw}) \times \left( e^{{Mu} + t} \right)}{\left( {{Yx}/{{sx}\lbrack{Feed}\rbrack}} \right)}$

[0162] where:

[0163] Mu=growth rate (in doublings per hour)

[0164] dcw=dry cell weight in grams per liter of medium at feed startdetermined empirically for individual E. coli strains

[0165] t=time in hours

[0166] Y x/s=glycerol utilization constant for host (0.327 for E. coliWA 837; similar for BLR)

[0167] v=fermentation volume in liters

[0168] [feed]=grams glycerol per liter of feed medium (629 grams perliter used in all examples reported herein).

[0169] Q=glycerol feed rate, liters/hour

[0170] The induction process is accomplished using a gradient inductioncoupled to the feed rate increasing the inducer (IPTG) over timematching the set growth rate.

[0171] The fermentation in progress is monitored by measuring absorbanceat 600 nm, wet cell weight in g/l, in addition to CEX chromatographyanalysis of media samples for the presence and concentration of sCTglyin mg/l.

[0172] Glycine can be added to the fermentation in order to increaseouter membrane permeability Glycine can be added to the batch media orto the feed. Preferably the glycine is added to the feed so that theglycine concentration in the fermentation culture increases with therate of cell growth. The optimal concentration of glycine should be inthe range of 0.1-1 grams per 100 ml at the final time point of thefermentation. In practice, the amount of glycine added to the feed iscalculated so that the desired glycine concentration at the fermentationend is achieved. The actual amount of glycine added is dependent on thelength of the fermentation in time, post induction, and final glycineconcentration desired. The methods we have used: 24 g/l of glycine wasadded to the fermentation feed, which results in a final glycineconcentration of 5 g/l at 26 hours post induction. We have found thatadding glycine to the feed is more effective than adding glycine to thebatch media. TABLE 2 Media Component List: Inoculation Media Batch MediaFeed Media Components Quantity g/L Components Quantity g/L ComponentsQuantity g/L (NH4)₂SO₄ 7.00 (NH4)₂SO₄ 14.80 Glycerol 629 KH₂PO₄ 2.00KH₂PO₄ 4.40 IPTG 2.80 MgSO₄—7H₂O 1.00 MgSO₄—7H₂O 2.10 CaCl₂ 0.25 CaCl₂0.53 FeSO₄—7H₂O 0.05 FeSO₄—7H₂O 0.11 Sodium Citrate 1.50 Sodium Citrate2.20 N-Z Case + 5.00 N-Z Case + 10.60 Hy Yest 412 2.00 Hy Yest 412 3.10L-Methionine 4.50 L-Methionine 4.50 Kanamycin 0.05 Kanamycin 0.05Glycerol 18.00 Glycerol 2.50

[0173] TABLE 3 Conditions determined for fermentation parameters MostParameter Preferred Preferred Good pH 6.78-6.85 6.50-7.00 6.00-7.5  Temp° C. 29.5-30.5 28.0-32.0 20.0-35.0 DO (oxygen saturation) >80% >70% >50%Mu value 0.12-0.14 0.10-0.16 0.05-0.20 Fermentation Time 24-27 22-2920-32 (Hours post induction)

[0174] Isolation of sCTglv

[0175] The conditioned medium is harvested by separating the cells frommedium using either Tangential Flow Filtration or centrifugation tocollect the media, and discarding the cells. The excreted sCTgly isstabilized in the media by adding 2.0 N HCl to a final pH of 3.0. Theglycine-extended salmon calcitonin is stable for extended periods oftime at pH 3.0. After cell removal and pH stabilization, the peptide ispurified using cation exchange and reverse-phase chromatography methods.While reverse phase chromatography followed by cation exchangechromatography can provide good purification, it is preferred that aninitial cation exchange step also be included prior to the reverse phaseliquid chromatography. For large purification, this reduces the volumeto be subjected to reverse phase chromatography, thus reducingenvironmental and safety concerns raised by the necessity of using highvolumes of organic solvents such as acetonitrile.

[0176] Another preferred modification is S-sulfonation of the cysteineresidues of the salmon calcitonin peptide prior to or duringpurification in order to improve yields of the monomeric peptide.

[0177] Description of Clones

[0178] The monogenic UGL 172 clone is an E. coil BLR host straincontaining vector pSCT-025A which comprises one transcription cassette(monogenic) coding for salmon calcitonin with a C-terminal glycine(sCTgly).² The digenic UGL 165 clone is an E. coli BLR host straincontaining vector pSCT-029A which comprises two cassettes in tandem(digenic) each coding for salmon calcitonin with a C-terminal glycine(sCTgly). The trigenic UGL 168 clone is an E. coli BLR strain containingvector pSCT-034 which comprises three cassettes in tandem (trigenic)each coding for salmon calcitonin with a C-terminal glycine (sCTgly).The monogenic UGL 702 clone is an E. coli BLR strain containing vectorpSCT-037 which comprises 1 cassette and secretion factor genes. Thedigenic UGL 703 clone is an E. coli BLR strain containing vectorpSCT-038 which comprises 2 cassettes in tandem and secretion factorgenes.

EXAMPLE 1 UGL 165 Fermentation at 1L Scale

[0179] The fermentation of the UGL 165 clone was carried out asdescribed under Experimental details. Table 4 summarizes thefermentation parameters and results. Briefly, UGL 165 clonal cells weregrown in inoculation medium and used to seed a fermenter containing 1liter of batch medium to give an initial A₆₀₀ nm of 0.06. Cells weregrown for 6.25 hours until the glycerol in the medium was depleted.Then, the fed batch stage of the fermentation was started andsupplemented continuously with the feed medium for 25.5 hours. Theconditions at time zero (beginning of feed and induction) were asfollows: oxygen saturation, 94%; temperature 30° C.; and pH 6.8. Theconditions at the end of fermentation (time 25.5 hours) were as follows:oxygen saturation, 40%; temperature 31° C.; and pH 6.8. Also, at the endof fermentation, the absorbance at 600 nm was equal to 113.3 and the wetcell weight in g per liter was 168 g. The sCTgly production at the endof fermentation was also measured to be 222 mg/liter of medium (see FIG.10 and table 4). TABLE 4 Fermentation Summary of UGL 165 Time point Hrs.wet cell sCT gly Feed vol Post A₆₀₀ weight mg/L added, [IPTG] AgitationFeed nm g/L ** mls uM RPM −6.25 0.06 — — — —  500  0*** 11.1 — — — —1300 15 51.7 83.3 not 44.1 528 1000 assayed 17 48.7 92.3 not 51.3 6141200 assayed 19 53.6 100.5 38 78.2 948 1300 21 67.6 117.3 77 104.8 12561300 22 86.7 130.9 111 120.4 1442 1350 23 92.6 140.0 104 138.2 1654 135024 106.5 153.7 156 158.4 1896 1350 25 108.5 162.8 183 181.2 2174 140025.5 113.3 168 222 194.1 2324 1400

EXAMPLE 2 UGL 703 Fermentation at 1 L Scale

[0180] Recombinant E. coli UGL 703 has been deposited with the AmericanType Culture Collection (ATCC) as ATCC 98395 in accordance with theprovision of the Budapest Treaty relating to the deposit ofmicroorganisms for purposes of patent procedure. The fermentation of theUGL 703 clone was carried out as described under Experimental details.Table 5 summarizes the conditions of this fermentation. Briefly, UGL 703clone was grown in inoculation medium and used to seed a fermentercontaining 1 liter batch medium to give an initial A₆₀₀ nm of 0.06(preference is 0.06 to 0.12). Cells were grown for 6.25 hours (preferredrange is from 6.0 to 7.0 hours) until the glycerol in the medium wasdepleted. Then, the fed batch stage of the fermentation was started andsupplemented continuously with the feed medium for 26 hours. Theconditions at time zero (beginning of feed and induction) were asfollows: oxygen saturation, 95%; temperature 30° C.; and pH 6.8. Theconditions at the end of fermentation (time 26 hours) were as follows:oxygen saturation, 80%; temperature 31° C.; and pH 6.8. Also, at the endof fermentation, the absorbance at 600 nm was equal to 80.9 and the wetcell weight in grams per liter was 129.1. The sCTgly production was alsomeasured to be 284 mg/liter of medium (see FIG. 11 and table 5). TABLE 5Fermentation Summary of UGL 703 wet cell sCT gly Feed vol Time weightmg/L added, [IPTG] Agitation point A 600 g/L ** mls uM RPM −6.5 0.06 — —— —  500 0 8.1 — — — — 1300 15.5 22.6 7.3 ˜18 47.5 570 1150 19 36.5 83.125 72.9 948 1250 21 53.5 94.2 53 104.8 1256 1350 22 61.3 100.8 58 120.41442 1450 23 59.7 106.0 69 138.2 1654 1550 24 73.3 119.0 83 158.4 18981580 25 74.1 119.1 203 181.1 2174 1620 26 80.9 129.1 284 207.6 2487 1620

[0181] Conclusions

[0182] Other experiments were carried out under similar overallconditions using UGL 172, UGL 168 and UGL 702 clones. FIGS. 12 and 13indicate that the digenic UGL 165 clone is best suited for production ofsCTgly with the trigenic UGL 165 clone being second best over themonogenic UGL 173 clone. However, the production of sCTgly by UGL 165can still be improved in the presence of co-expressed secretion factors(UGL 703) (compare FIGS. 10 and 11 and 17).

[0183] With regard to oxygen saturation during fermentation, FIGS. 14Aand 14B support the conclusion that added oxygen in the fermentationmedium is not critical to cell growth of UGL 165 but is very importantin increasing the production of sCTgly.

[0184]FIGS. 15A, 15B, and 16 clearly indicate that the E. coli strainBLR is best suited for production of sCTgly.

[0185] The production of sCTgly can be still further increased by theaddition of glycine as an added feed component.

EXAMPLE 3 Purification of sCTgly from UGL 165 Culture

[0186] Media: Cation-exchange Chromatography #1:

[0187] Approximately 1000 L of culture media which had been harvested byeither tangential flow filtration or centrifugation was acidified with asufficient volume of 2N hydrochloric acid to decrease the pH to 3.0. Themedia was subsequently diluted with a sufficient volume of water todecrease the conductivity to <7.5 mS. The diluted media was loaded ontoa cation-exchange column (Pharmacia SP-Sepharose Big Beads, 99.0 cm×13.0cm) which had been equilibrated with 10 mM citric acid pH 3.0 at a flowrate of 25 L/min. (3.25 cm/min.). After the loading was complete, thecolumn was washed with 10 mM citric acid pH 3.0 at 8 L/min (1.0 cm/min)for approximately 40 minutes (3 bed volumes) or until a stable UVbaseline was achieved. The product (sCTgly) was eluted with 10 mM citricacid, 350 mM sodium chloride pH 3.0 at a flow rate of 8 L/min. (1.0cm/min.). The column was cleaned and sanitized with 0.5 M sodiumhydroxide. 1.0 M sodium chloride for 60.0 minutes (5.0 bed volumes) at 8L/min. (1.0 cm/min).

[0188] To a stirred tank containing the resulting CEX#L eluate(approximately 100 L) is added 60.57 grams of tromethamine. The solutionis stirred until all solids are dissolved. The pH of the solution isadjusted to 8.25 [range: 8.0 to 8.5] using 2 M NaOH. 23.64 grams ofTRIS·HCl (Tris[hydroxymethyl]aminomethane hydrochloride) is added andthe solution is stirred until all solids are dissolved. A solution of1.0 kg of sodium sulfite dissolved in TRIS·HCl and a solution of 200grams of sodium tetrathionate dissolved in TRIS·HCl are added to thetank with stirring. The reaction is allowed to stir for 15 minutes. Ifnecessary, the pH is adjusted to 8.25 [range: 8.0 to 8.5] with 2 M NaOH.The reaction mixture is stirred overnight at room temperature. The pH ofthe reaction mixture is adjusted to 2.25 [range: 2.0 to 2.5] with 2 MHCl.

[0189] Reverse-phase Chromatoaraphy #1 (RP #1):

[0190] The resulting S-sulfonation reaction mixture (approximately 100L) was loaded directly onto a reverse-phase column (Toso Haas AmberchromCG300 md, 25.0 cm×18.0 cm) which had been equilibrated with 0.1% trifluoroacetic acid at 2.0 L/min. (4.0 cm/min). After loading was complete,the column was washed with 0.1% trifluoroacetic acid at 750 ml/min. (1.5cm/min.) until a stable UV baseline was achieved. The column was washedwith 0.1% trifluoroacetic acid, 20% acetonitrile at 750 ml/min. (1.5cm/min.) until the principal contaminant peak completely eluted. Theproduct (sCTgly) was eluted with 0.1% trifluoroacetic acid, 40%acetonitrile at 750 ml/min. (1.5 cm/min.). The column was cleaned with0.1% trifluoroacetic acid, 80% acetonitrile for 30 minutes at 750ml/min. (1.5 cm/min.).

[0191] Cation-exchange Chromatography #2 (CEX #2):

[0192] The resulting RP #1 eluate (approximately 8.0 L) was loadeddirectly onto a cation-exchange column (E. Merck Fractogel EMD S03 650M,18.0 cm×24.0 cm) which had been equilibrated with 25 mM MES(2-[N-morpholino]-ethanesulfonic acid) pH 5.8 at 500 ml/min. (2.0cm/min.). After the loading was complete, the column was washed at 750ml/min. (3.0 cm/min.) with 25 mM MES (2-[N-morpholino]-ethanesulfonicacid) pH 5.8 until the column effluent returned to pH 5.8 (range5.6-5.9). The column was washed with 25 mM MES(2-[N-morpholino]-ethanesulfonic acid), pH 5.8 for an additional 30minutes at 750 ml/min. (3.0 m/min.) to remove the principal peptidecontaminants. The product (sCTgly) was eluted with 25 mM MES, 100 mMsodium chloride pH 5.8 at 750 ml/min. (3.0 cm/min.). The productfraction is adjusted to pH 3.0-5.0 with 1.0 M HCl unless amidationfollows immediately. The column was cleaned and sanitized with 0.1 Msodium hydroxide, 1.0 M sodium chloride for 60 minutes at 750 ml/min.(3.0 m/min.).

[0193] Amidation Reaction.

[0194] The resulting pH-adjusted CEX #2 eluate contains purified sCTglywhich is a suitable substrate solution for use in the in vitroconversion of sCTgly to authentic salmon calcitonin, a reactioncatalyzed by peptidyl glycine a-amidating enzyme (PAM) as shown below inExample 5.

EXAMPLE 4

[0195] Analytical Cation-exchange HPLC for Quantification of sCTgly:

[0196] sCTgly in collected chromatography fractions was identified andquantified by analytical CEX-HPLC. An aliquot of each fraction wasloaded onto a cation-exchange column (The Nest Group, Polysulfoethylaspartamide, 4.6 mm×50 mm) which had been equilibrated with 10 mM sodiumphosphate pH 5.0 at a flow rate of 1.2 ml/min. Separation was achievedby performing a linear gradient from 10 mM sodium phosphate pH 5.0 to 10mM sodium phosphate, 250 mM sodium chloride pH 5.0 at 1.2 ml/min over 15minutes. The column effluent was monitored by UV absorbance at 220 nm.sCTgly was identified by comparison of its retention time to that of apurified sCTgly reference standard. sCTgly was quantified by peak areaas compared to the sCTgly reference standard. This analytical method wasalso used to quantify SCTgly from the fermentation medium.

EXAMPLE 5 Conversion of Glycine-extended Salmon Calcitonin to AuthenticSalmon Calcitonin using α-amidating Enzyme

[0197] In order to obtain the optimal yields of amidated salmoncalcitonin, the following critical parameters are observed:

[0198] 1) The amidation reaction is carried out in a silanized glassvessel to prevent non-specific adsorption of peptide to the reactionvessel.

[0199] 2) A high level of dissolved oxygen is maintained in the reactionmixture by sparging and/or agitation. Preferably, the level of dissolvedoxygen is >75%.

[0200] 3) Incubation temperature during amidation is maintained between35° C.-39° C.

[0201] 4) The pH of the amidation reaction is maintained between 6.0 and6.5.

[0202] 5) The starting concentration of glycine-extended salmoncalcitonin in the amidation reaction should be between 3.5-10.5 mg/ml(0.95 mM to 2.9 mM).

[0203] 6) When 12,000-24,000 units/ml of substantially protease-freea-amidating enzyme (peptidyl glycine a-amidating monooxygenase, hereinreferred to as “PAM”) are added to the reaction mixture and theconcentration of substrate is as indicated in 5) above, the reaction isallowed to proceed for 4-6 hours. However, the reaction time can befurther increased up to 24 hours without deleterious effects to theproduct.

[0204] 7) To prevent the amidation reaction from becoming ascorbatelimiting, an additional equivalent of ascorbate is added at about themidpoint of the reaction.

[0205] The components of the amidation reaction mixture are thefollowing:

[0206] 3.5-10.5 mg/ml of S-sulfonated, glycine extended salmoncalcitonin

[0207] 30 mM MES buffer, pH 6.0-6.5

[0208] 0.5 to 1.0 uM CUSO₄ (e.g. 0.5)

[0209] 4-15 mM KI (e.g. 5)

[0210] 1-5% Ethanol (e.g. 1%)

[0211] 10-100 ug/ml Catalase (e.g. 35)

[0212] 1.5-3.0 mM Ascorbate (e.g. 1.5)

[0213] peptidyl glycine α-amidating monooxygenase (12,000-24,000 unitsper ml of reaction mixture. 1 unit is 1 picomole per minute conversionof DansylTyr-Val-Gly substrate to product at 37° C. at pH 7). The PAMenzyme may be obtained as described in Miller et al., ABB 298: 380-388(1992) U.S. Pat. No. 4,708,934, European publication 0 308 067 and 0 382403, and Biotechnology Vol. II (1993) pp. 64-70, the disclosures ofwhich are hereby incorporated by reference.

[0214] The glycine extended salmon calcitonin may be produced by thefermentation as described in Example 1 or Example 2 and purified asdescribed in Example 3 prior to amidation.

[0215] In instances where the enzyme used for amidation is peptidylglycine α-hydroxylating monooxygenase (PHM), the same reaction mixtureis used as that described above, substituting PHM for PAM. In addition,at the end of the 4 to 6 hour incubation period, the pH of the reactionmixture is increased by the addition of base to between 8 and 9. Thereaction mixture is agitated for an additional 4 to 8 hours prior toterminating the reaction. Peptidyl glycine α-hydroxylating monooxygenasemay be obtained by expressing only the N-terminal portion of PAM (aboutthe first 40 dKa). See e.g. Mizuno et al. BBRC Vol. 148, No. 2, pp.546-52 (1987) the disclosure of which (as it relates to Mizuno's “AE 1”is incorporated herein by reference Frog skin is known to express PHMnaturally.

[0216] After the amidation reaction has been terminated, the reaction isdiluted with sufficient water to bring the final peptide concentrationto less than 3.0 mg/ml. Sufficient 1 M TRIS pH 9.0 is added to themixture to bring the final concentration of TRIS to approximately 100mM. If necessary, the pH is adjusted to [8.0 to 9.0] with 2 M NaOH. A3.0 fold excess of L-cysteine, over the final concentration (mM) ofSO₃-sCT, is added slowly with stirring to the reaction mixture. Ifnecessary, the pH is adjusted to [8.0 to 8.5] with 2 M NaOH. Therenaturation reaction is stirred for 1 hour at room temperature. Thereaction is terminated by acidification with 10% phosphoric acid to pH2.0 [1.9 to 2.3].

EXAMPLE 6

[0217] Post-amidation Purification

[0218] Cation Exchange Chromatography #3 (CEX #3):

[0219] This column is used to purify sCT following α-amidation andrenaturation. The principal contaminant followingα-amidation/renaturation is SCTG. CEX t3 chromatography employs anAmicon Vantage-A column (18.0×16.0 cm) packed with Toyopearl SP650Sresin. The unit operation is accomplished using water for injection(WFI) and solutions of 0.5 M, 50 mM and 175 mM sodium chloride alongwith 150 mM sodium phosphate pH 5.5. A brief description of the processsteps follows:

[0220] 1) The operational flow rate is set to 750 ml/min:

[0221] 2) The following parameters which are used to monitor thechromatography are set using LC system controller:

[0222] UV wavelength 230 nm

[0223] Range 0.64 AUFS

[0224] Conductivity×1000

[0225] 3) The column is initially washed with WFI for at least 5 minutesat a flow rate of 750 ml/min.

[0226] 4) The dilution pump (150 mM sodium phosphate pH 5.0) is set to50 ml/min. and the column is equilibrated with 10 mM sodium phosphate ata flow rate of 750 ml/min until a stable pH baseline is observed.

[0227] 5) The column is re-equilibrated with 10 mM sodium phosphate at aflow rate of 750 ml/min until a stable pH baseline below 6.0 isachieved. (Note: If the pH of the column is not below 6.0 then a 150 mMsodium phosphate wash is required.) If the 150 mM sodium phosphate pH5.5 wash is performed the column must be re-equilibrated using 10 mMsodium phosphate pH 5.5 before proceeding at the next step.

[0228] 6) Following the re-equilibration, the column is subjected to ablank elution with 175 mM sodium chloride; 10 mM sodium phosphate pH 5.5for 4 minutes at a flow rate of 750 ml/min.

[0229] 7) The column is re-equilibrated with 10 mM sodium phosphate pH5.5 at a flow rate of 750 ml/min until a stable pH baseline is achieved.

[0230] 8) Once equilibration is achieved the amidated/renatured outputcontaining 10-25 gram of sCT is pH adjusted to 3.5 using 2 N sodiumhydroxide and loaded onto the CEX #3 column at 400 ml/min. The sampleload is chased by rinsing the load container with 500 ml of WFI.

[0231] 9) Following the load the column is washed with 10 mM sodiumphosphate pH 5.5 at a flow rate of 750 ml/min for 30 minutes or untilthe pH of the column stabilizes above 5.0.

[0232] 10) Once the pH of the column has stabilized above 5.0 the columnis washed, at 750 ml/min., with 50 mm sodium chloride; 10 mm sodiumphosphate pH 5.5 for 100 minutes or until the sCTgly peak emerges.

[0233] 11) Once the 100 minutes has expired the 175 mm sodium chlorideis attached to the system. The column is washed with 175 mM sodiumchloride; 10 mM sodium phosphate pH 5.5 at a flow rate of 750 ml/min.and the product eluted. The entire product peak is collected in onecontainer. The weight of the CEX3 output material is determined and 1 Nacetic acid (10% fraction weight) is added to the fraction.

[0234] 12) The column is stripped with 0.5 M sodium chloride; 10 mMsodium phosphate pH 5.5 at a flow rate of 750 ml/min. for 15 minutes.

[0235] 13) Once the column has been stripped with 1.0 M sodiumchloride/0.25 N sodium hydroxide is attached to the system. The dilutionpump is set to 0.000 ml/min. and the column washed with 1.0 M sodiumchloride/0.25 N sodium hydroxide at a flow rate of 600 ml/min. for atleast 30 minutes.

[0236] 14) The column is washed with WFI for 5 minutes at a flow rate of750 ml/min.

[0237] 15) The column is washed with 10 mM sodium hydroxide at a flowrate of 500 ml/min. for at 30 minutes. The column is stored under theseconditions.

[0238] Reverse-phase Chromatography (RP #2):

[0239] This step follows CEX #3 and is used as a salt and bufferexchange step prior to lyophilization. The main objective of the step isto exchange the salt with acetate. RP #2 chromatography employs anAmicon Vantage-A column (13.0×12.5 cm) packed with Amberchrom CG300mdresin. The unit operation is accomplished using water for injection,ethyl alcohol, 250 mM sodium acetate and 0.5% acetic acid. A briefdescription of the process steps follows:

[0240] 1) The CEX #3 eluate (approximately 4 liters) is acidified to pH2.0 with phosphoric acid and then diluted with 3 equal volumes of 333 mMsodium acetate solution, and allowed to stand for at least 1 hour.

[0241] 2) The flow rate is set to 320 ml/min. while the dilution pump(0.5% acetic acid) is set to 80 ml/min. for an overall operational flowrate of 400 ml/min.

[0242] 3) The following parameters which are used to monitor thechromatography are set using LC system controller:

[0243] UV wavelength 230 nm

[0244] Range 2.54 AUFS

[0245] Conductivity×1000

[0246] 4) The column is initially washed with 0.1% acetic acid at a flowrate of 400 ml/min. until a stable conductivity baseline is observed.

[0247] 5) The column is stripped with 80% ethyl alcohol, 0.1% aceticacid at a flow rate of 400 ml/min. until a stable pH baseline isobserved.

[0248] 6) The column is washed with 0.1% acetic acid until a stable pHbaseline is observed.

[0249] 7) Following the wash, a column test is performed to track resincleaning. The column is subjected to a blank elution with 40% ethylalcohol; 0.1% acetic acid for 6 minutes at a flow rate of 400 ml/min.The collected eluate from the column test is submitted at QC foranalytical testing.

[0250] 8) The column is washed with 0.1% acetic acid at a flow rate of400 ml/min until a stable conductivity baseline is observed.

[0251] 9) Upon completion of the wash, the WFI is disconnected from theinlet and the 250 mM sodium acetate is connected. The dilution pump isset to 0.000 ml/min. The column is equilibrated with 250 mM sodiumacetate at a flow rate of 400 ml/min. until a stable pH baseline isobserved.

[0252] 10) Once equilibration is achieved, the CEX #3 eluate is loadedonto the RP #2 column at 400 ml/min. The sample load is chased byrinsing the load container with 1.0 liters of 250 mM sodium acetate.

[0253] 11) The column is washed using 250 mM sodium acetate at a flowrate of 400 ml/min. for 60 minutes.

[0254] 12) Following the sodium acetate wash, the sodium acetate isdisconnected from the inlet and the WFI is connected. The dilution pumpis returned to a flow rate of 80 ml/min. and the column is washed with0.1% acetic acid at a flow rate of 400 ml/min for 25 minutes.

[0255] 13) Following the 0.1% acetic acid wash, the product is elutedusing 40% ethyl alcohol; 0.1% acetic acid at a flow rate of 400 ml/min.The entire product peak is collected, and subjected to lyophilization toyield purified sCT powder.

[0256] 14) The column is stripped with 80% ethyl alcohol; 0.1% aceticacid at a flow rate of 400 ml/min. for at least 20 minutes.

[0257] 15) Following the strip, the dilution pump is set to 0.000ml/min. and the column is washed with WFI at a flow rate of 400 ml/min.for at least 5 minutes.

[0258] 16) After the WFI wash, the WFI is disconnected from the inletand the 0.5N sodium hydroxide is connected. The column is washed withthe 0.5N sodium hydroxide at a flow rate of 400 ml/min. for at least 20minutes.

[0259] 17) The 0.5N sodium hydroxide is disconnected from the system andthe WFI is connected. The column is washed with 50% ethyl alcohol at aflow rate of 400 ml/min. for at least 20 minutes. The column is storedunder these conditions.

[0260] 18) The RP #3 eluate is stored at 2 to 8° C.

[0261] Although the present invention has been described in relation toparticular embodiments thereof, many other variations and modificationsand other uses will become apparent to those skilled in the art. Thepresent invention therefore is not limited by the specific disclosureherein, but only by the appended claims.

What is claimed is:
 1. An expression vector comprising: (a) a codingregion with nucleic acids coding for a peptide product coupled inreading frame 3′ of nucleic acids coding for a signal peptide; and (b) acontrol region linked operably with the coding region, said controlregion comprising a plurality of promoters and at least one ribosomebinding site, wherein at least one of said promoters is tac.
 2. Thevector of claim 1, comprising a plurality of transcription cassettes,each cassette having said control region and said coding region.
 3. Thevector of claim 1, further comprising nucleic acids coding for arepressor peptide capable of repressing expression controlled by atleast one of said promoters.
 4. The vector of claim 3, wherein thenucleic acids coding for the repressor encode a lac repressor.
 5. Thevector of claim 1, wherein said control region has exactly twopromoters.
 6. The vector of claim 1, wherein said tac promoter is 5′ ofanother promoter in said control region.
 7. The vector of claim 1,wherein the control region comprises both a tac promoter and a lacpromoter.
 8. The vector of claim 7, wherein the lac promoter is 3′ ofthe tac promoter.
 9. The vector of claim 1, wherein said nucleic acidscoding for the signal peptide encode a signal peptide for secretedbacterial proteins.
 10. The vector of claim 9, wherein said signal isOmpA signal peptide.
 11. The vector of claim 1, wherein said peptideproduct has a molecular weight of less than 10 KDa.
 12. The vector ofclaim 1, wherein the C-terminal amino acid of said peptide product isglycine.
 13. The vector of claim 12, wherein said peptide product issalmon calcitonin precursor.
 14. The vector of claim 12, wherein saidpeptide product is calcitonin gene related peptide precursor.
 15. Thevector of claim 12, wherein said peptide product is selected from thegroup consisting of parathyroid hormone, the first 34 amino acids ofparathyroid hormone, and a 35 amino acid peptide having a C-terminalglycine in position 35 and the first 34 amino acids of parathyroidhormone in positions 1-34.
 16. The vector of claim 1, further comprisingnucleic acids coding for at least one secretion enhancing peptide. 17.The vector of claim 16, wherein the secretion enhancing peptide isselected from the group consisting of secY and prlA-4 .
 18. The vectorof claim 16, wherein the secretion enhancing peptide is secE.
 19. Thevector of claim 16, wherein a plurality of secretion enhancing peptidesare encoded, at least one of which is secE and the other of which isselected from the group consisting of secY and prlA-4.
 20. A host celltransformed or transfected with the vector of claim
 1. 21. A host celltransformed or transfected with the vector of claim
 16. 22. The hostcell of claim 20, wherein said host cell is a bacterial cell.
 23. Thehost cell of claim 22, wherein said bacterial cell is a gram negativebacterial cell.
 24. The host cell of claim 22, wherein said bacterialcell is E. coli.
 25. The host cell of claim 24, wherein said E. coli isstrain BLR.
 26. The host cell of claim 24, wherein said E. coli isstrain BL21.
 27. The host cell of claim 24, wherein said E. coil isstrain WA837.
 28. The host cell of claim 22, wherein said host cellfurther expresses at least one secretion-enhancing peptide.
 29. The hostcell of claim 28, wherein the secretion enhancing peptide is selectedfrom the group consisting of secY and prlA-4.
 30. The host cell of claim28, wherein the secretion enhancing peptide is secE.
 31. The host cellof claim 28, wherein a plurality of secretion enhancing peptides areencoded, at least one of which is secE and the other of which isselected from the group consisting of secY and prlA-4.
 32. An E. coilhost containing and expressing an expression vector which comprises aplurality of transcription cassettes in tandem, each cassettecomprising: (a) a coding region comprising nucleic acids coding for apeptide product coupled in reading frame 3′ of nucleic acids coding fora signal peptide; and (b) a control region linked operably with thecoding region, said control region comprising a plurality of promotersin tandem and at least one ribosome binding site, wherein at least oneof said promoters is selected from the group consisting of tac and lac.33. The host of claim 32, wherein said control region comprises from 5′to 3′ a tac promoter and a lac promoter.
 34. The host of claim 32,wherein said vector has exactly two transcription cassettes in tandem.35. A host cell transformed with an expression vector which comprises agene for expressing salmon calcitonin precursor, said host cell being E.coli strain BLR.
 36. A host cell transformed with an expression vectorwhich comprises a gene for expressing calcitonin gene related peptideprecursor, said host being E. coli strain BLR.
 37. A method of producinga peptide product which comprises culturing the host cell of claim 20 ina culture medium and then recovering the peptide product from the mediumin which the host cell has been cultured.
 38. The method of claim 37wherein peptide product yield exceeds 100 mg per liter of media.
 39. Amethod of producing a peptide product which comprises culturing the hostcell of claim 21 in a culture medium and then recovering the peptideproduct from the medium in which the host cell has been cultured.
 40. Amethod of producing a peptide product which comprises culturing the hostcell of claim 35 in a culture medium and then recovering the peptideproduct from the medium in which the host cell has been cultured. 41.The method of claim 37, wherein the peptide product is salmon calcitoninprecursor.
 42. The method of claim 37, wherein the peptide product iscalcitonin gene related peptide precursor.
 43. The vector of claim 1,wherein said peptide product is selected from the group consisting ofparathyroid hormone, the first 34 amino acids of parathyroid hormone and35 amino acid peptide having a C-terminal glycine in position 35 and thefirst 34 amino acids of parathyroid hormone in positions 1-34.
 44. Themethod of claim 38, wherein a method of induction is started prior tostationary phase.
 45. The method of claim 44, wherein the inductionmethod is by addition of a chemical inducer.
 46. The method of claim 45,wherein the induction is by addition of at least one inducer selectedfrom the group consisting of IPTG and lactose.
 47. A method of producinga peptide product which comprises the steps of: (a) culturing host cellsof claim 20 in a culture medium in the presence of a carbon source andinducing the expression of the peptide product, while controlling growthof said host cells at a growth rate between 0.05 and 0.20 doublings perhour; and (b) thereafter recovering said peptide product from themedium.
 48. The method of claim 47, wherein a membrane-permeabilizingamount of glycine is present in the medium during at least a portion ofsaid controlled growth.
 49. The method of claim 47, wherein an inducerand carbon source are added during each hour of culturing in amountssuch that the weight ratio of the inducer to the carbon source added inany one hour does not vary by more than 50% from the ratio added duringthe entire fermentation period.
 50. The method of claim 47, wherein noexternal carbon source is introduced into the medium until carbon sourceinitially present in said medium is depleted to a level at which itcould not continue to support the life of said host absent introductionof external carbon source into the medium, and wherein carbon source isthereafter added at a rate which maintains said growth rate between 0.05and 0.20 doublings per hour.
 51. The method of claim 47, wherein thehost cell is cultured for a period between 20 and 32 hours postinduction.
 52. The method of claim 47, wherein the host cell is culturedfor a period between 24 and 27 hours post induction.
 53. The method ofclaim 47, wherein the host cell is cultured at a temperature between 20and 35° C.
 54. The method of claim 47, wherein the host cell is culturedat a temperature between 28 and 32° C.
 55. The method of claim 47,wherein the host cell is cultured at a temperature between 29.5 and30.5° C.
 56. The method of claim 47, wherein the pH of the culturemedium is between 6.0 and 7.5.
 57. The method of claim 47, wherein thepH of the culture medium is between 6.78 and 6.85.
 58. The method ofclaim 47, wherein the pH of the culture medium is between 6.6 and 7.0.59. The method of claim 47, wherein oxygen saturation of the culturemedium is at least 20%.
 60. The method of claim 47, wherein oxygensaturation of the culture medium is at least 50%.
 61. The method ofclaim 47, wherein the oxygen saturation of the culture medium is atleast 80%.
 62. The method of claim 46, wherein the average cell growthrate during the culture period is maintained between 0.10 and 0.15doublings per hour.
 63. The method of claim 55, wherein the growth rateis maintained at about 0.13 doublings per hour.
 64. The method of claim49, wherein the carbon source is glycerol.
 65. The method of claim 37,wherein said peptide product is salmon calcitonin precursor.
 66. Themethod of claim 37, wherein said peptide product is calcitonin generelated peptide precursor.
 67. The method of claim 37, wherein saidpeptide product is selected from the group consisting of parathyroidhormone, the first 34 amino acids of parathyroid hormone and 35 aminoacid peptide having a C-terminal glycine in position 35 and the first 34amino acids of parathyroid hormone in positions 1-34.
 68. The method ofclaim 47, wherein induction is achieved by use of a chemical inducer.69. The method of claim 58, wherein the inducer is at least one agentselected from the group consisting of lactose and IPTG.
 70. The methodof claim 37, wherein recovering said peptide product comprises: (a)separating host cells from the culture medium; and (b) subjecting themedium to reverse-phase liquid chromatography and recovering fractionscontaining peptide product; and (c) subjecting said fractions of step(b) to cation exchange chromatography, and (d) thereafter recoveringfractions containing peptide product.
 71. The method of claim 39,wherein recovering said peptide product comprises: (a) separating hostcells from the culture medium; and (b) subjecting the medium toreverse-phase liquid chromatography and recovering fractions containingpeptide product; and (c) subjecting said fractions of step (b) to cationexchange chromatography, and (d) thereafter recovering fractionscontaining peptide product.
 72. The method of claim 47, whereinrecovering said peptide product comprises: (a) separating host cellsfrom the culture medium; and (b) subjecting the medium to reverse-phaseliquid chromatography and recovering fractions containing peptideproduct; and (c) subjecting said fractions of step (b) to cationexchange chromatography, and (d) thereafter recovering fractionscontaining peptide product.
 73. The method of claim 37, whereinrecovering said peptide product comprises: (a) separating host cellsfrom the culture medium; and (b) subjecting the medium to cationexchange chromatography and recovering fractions containing said peptideproduct; and (c) subjecting the recovered fraction of step (b) toreverse-phase liquid chromatography and recovering fractions containingpeptide product; (d) subjecting the recovered fractions of step (c) tocation exchange chromatography, and (e) thereafter recovering fractionscontaining peptide product.
 74. The method of claim 39, whereinrecovering said peptide product comprises: (a) separating host cellsfrom the culture medium; and (b) subjecting the medium to cationexchange chromatography and recovering fractions containing said peptideproduct; and (c) subjecting the recovered fraction of step (b) toreverse-phase liquid chromatography and recovering fractions containingpeptide product; (d) subjecting the recovered fractions of step (c) tocation exchange chromatography, and (e) thereafter recovering fractionscontaining peptide product.
 75. The method of claim 47, whereinrecovering said peptide product comprises: (a) separating host cellsfrom the culture medium; and (b) subjecting the medium to cationexchange chromatography and recovering fractions containing said peptideproduct; and (c) subjecting the recovered fraction of step (b) toreverse-phase liquid chromatography and recovering fractions containingpeptide product; and (d) subjecting the recovered fractions of step (c)to cation exchange chromatography, and (e) thereafter recoveringfractions containing peptide product.
 76. The method of claim 70,wherein the peptide product has at least one cysteine in its molecularstructure and wherein at least one sulfhydryl group of a cysteine of thepeptide product is sulfonated during at least a portion of said methodof recovering peptide product.
 77. The method of claim 71, wherein thepeptide product has at least one cysteine in its molecular structure andwherein at least one sulfhydryl group of a cysteine of the peptideproduct is sulfonated during at least a portion of said method ofrecovering peptide product.
 78. The method of claim 72, wherein thepeptide product has at least one cysteine in its molecular structure andwherein at least one sulfhydryl group of a cysteine of the peptideproduct is sulfonated during at least a portion of said method ofrecovering peptide product.
 79. The method of claim 73, wherein thepeptide product has at least one cysteine in its molecular structure andwherein at least one sulfhydryl group of a cysteine of the peptideproduct is sulfonated during at least a portion of said method ofrecovering peptide product.
 80. The method of claim 74, wherein thepeptide product has at least one cysteine in its molecular structure andwherein at least one sulfhydryl group of a cysteine of the peptideproduct is sulfonated during at least a portion of said method ofrecovering peptide product.
 81. The method of claim 75, wherein thepeptide product has at least one cysteine in its molecular structure andwherein at least one sulfhydryl group of a cysteine of the peptideproduct is sulfonated during at least a portion of said method ofrecovering peptide product.
 82. The method of claim 72, furthercomprising altering the pH of the culture medium, immediately afterterminating fermentation, to a level where proteolytic degradation ofproduct is reduced.
 83. The method of claim 82, wherein salmoncalcitonin precursor is the peptide product and pH is adjusted tobetween 2.5 and 4.0.
 84. The method of claim 83, wherein the pH isadjusted to between 3 and 3.5.
 85. The method of claim 82, furthercomprising lowering the temperature of the culture medium to below 10°C. after fermentation is terminated.
 86. A method of producing anamidated peptide product comprising the steps of: (a) culturing the hostcell of claim 20 in a culture medium wherein the peptide productincludes a C-terminal glycine; (b) recovering said peptide product fromsaid culture medium; and (c) converting said peptide product to anamidated peptide by converting said C-terminal glycine to an aminogroup.
 87. A method of producing an amidated peptide product comprisingthe steps of: (a) culturing the host cell of claim 35 in a culturemedium wherein the peptide product includes a C-terminal glycine; (b)recovering said peptide product from said culture medium; and (c)converting said peptide product to an amidated peptide by convertingsaid C-terminal glycine to an amino group.
 88. A method of producing anamidated peptide comprising: (a) culturing host cells which express apeptide product having a C-terminal glycine together with an N-terminalsignal peptide under conditions wherein growth of said host cells iscontrolled to stay within a range of 0.05 to 0.20 doublings per hour;wherein the culture is induced during some of the period of saidcontrolled growth; (b) recovering said peptide product from the culturemedia; and (c) converting said peptide product to an amidated peptide byconverting said C-terminal glycine to an amino group.
 89. The method ofclaim 83, wherein the host cell is cultured in a culture medium in thepresence of an inducer, while maintaining an average cell growth rateduring culturing between 0.05 and 0.20 doublings per hour.
 90. Themethod of claim 84, wherein the host cell is cultured in a culturemedium in the presence of an inducer, while maintaining an average cellgrowth rate during culturing between 0.05 and 0.20 doublings per hour.91. The method of claim 86, wherein the peptide product is salmoncalcitonin precursor or calcitonin gene related peptide precursor. 92.The method of claim 87, wherein the peptide product is salmon calcitoninprecursor or calcitonin gene related peptide precursor.
 93. The methodof claim 86, wherein said conversion to amidated peptide is accomplishedby: (a) forming a reaction mixture by contacting said peptide productwith oxygen and a reducing agent in the presence of peptidyl glycineα-amidating monooxygenase, or peptidyl glycine α-hydroxylatingmonooxygenase; (b) if peptidyl glycine α-amidating monooxygenase is notused in step (a), and if the reaction mixture is not already basic, thenincreasing pH of the reaction mixture until it is basic; and (c)recovering said amidated peptide from said reaction mixture.
 94. Themethod of claim 87, wherein said conversion to amidated peptide isaccomplished by: (a) forming a reaction mixture by contacting saidpeptide product with oxygen and a reducing agent in the presence ofpeptidyl glycine α-amidatlng monooxygenase, or peptidyl glycineα-hydroxylating monooxygenase; (b) if peptidyl glycine α-amidatingmonooxygenase is not used in step (a), and if the reaction mixture isnot already basic, then increasing pH of the reaction mixture until itis basic; and (c) recovering said amidated peptide from said reactionmixture.
 95. The method of claim 88, wherein said conversion to amidatedpeptide is accomplished by: (a) forming a reaction mixture by contactingsaid peptide product with oxygen and a reducing agent in the presence ofpeptidyl glycine α-amidating monooxygenase, or peptidyl glycineα-hydroxylating monooxygenase; (b) if peptidyl glycine α-amidatingmonooxygenase is not used in step (a), and if the reaction mixture isnot already basic, then increasing pH of the reaction mixture until itis basic; and (c) recovering said amidated peptide from said reactionmixture.
 96. The method of claim 93, wherein recovering amidated peptidecomprises at least one of the steps selected from the group consistingof cation exchange chromatography and reverse phase chromatography. 97.The method of claim 94, wherein recovering amidated peptide comprises atleast one of the steps selected from the group consisting of cationexchange chromatography and reverse phase chromatography.
 98. The methodof claim 84, wherein recovering amidated peptide comprises at least oneof the steps selected from the group consisting of cation exchangechromatography and reverse phase chromatography.
 99. A method for directexpression of a peptide product into a culture medium comprising thesteps of: (a) culturing host cells which express said peptide producttogether with a signal peptide, in said medium, under conditions whereingrowth of said host cells is controlled to stay within a range of 0.05to 0.20 doublings per hour; wherein an inducer is present during some ofsaid period of controlled growth; and (b) recovering said peptideproduct from the culture medium.
 100. The method of claim 99, wherein amembrane-permeabilizing amount of glycine is present in the mediumduring at least a portion of said period of controlled growth.
 101. Themethod of claim 99 wherein oxygen saturation averages over 50% in saidmedium during the period of controlled growth.